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Dynoles cause cytokinesis failure by blocking completion of abscission. A, schematic of experimental design. G2–M synchronized cells (RO-3306) were released into either drug-free medium or medium containing dynamin inhibitors and analyzed at 6 hours for multinucleation or over 20-hour period by using time-lapse microscopy. B, synchronized HeLa cells were treated with each dynole (10 μmol/L) or MiTMAB (10 μmol/L) as described in A. At 6 hours, cells were fixed, stained for α-tubulin, and the percentage of cells that were multinucleated were scored by using immunofluorescence microscopy. Bar graph is mean ± SEM, n = 3. C–F, synchronized HeLa cells were treated with vehicle (0.1% DMSO) or each dynole (10 μmol/L) as described in A, then visualized by time-lapse microscopy. Cells entering mitosis were scored for the time taken to progress through mitosis from prophase (Pro) to completion of mitosis (Comp) or generation of a binucleated cell (Multi; C). Cells were also scored for the time taken to progress through the following critical mitotic phases: metaphase (Met) to anaphase (Ana; D), Ana to full membrane ingression (Ing; E), and Ing to Comp or Multi (F). *, P < 0.05, Student's t test. G and H, representative time-lapse images of HeLa cells treated with DMSO and 10 μmol/L Dynole 34-2 undergoing mitosis. DMSO-treated cells (G) completed abscission generating 2 independent cells. Dynole 34-2–treated cells (H) failed cytokinesis by failing to abscise the ICB. The cleavage furrow regressed, forming a binucleated cell. Percentage of cells that entered mitosis and failed cytokinesis are shown below.

Cell death following cytokinesis failure in HeLa cells exposed to dynole 34-2. A, HeLa cells were treated and visualized as described in Fig. 1A. Percentage of cells that underwent apoptosis following cytokinesis failure and following a successful mitotic division is shown (mean ± SD, n = 2). B, representative time-lapse images of a HeLa cell treated with dynole 34-2 or 0.1% DMSO alone. Dynole 34-2–treated cells undergo apoptotic cell death, characterized by membrane blebbing, 125 minutes after failing cytokinesis. C and D, HeLa cells were treated as described in A except that after 20 hours, the DNA content was analyzed by flow cytometry. Percentage of cells (mean ± SD, n = 2) with less than 2N (C) and 4N DNA content (D) are shown. *P < 0.05, Student's t tests. E, HeLa cells were treated as described in A. At 8 hours, lysates were immunoblotted for cleaved PARP. γ-tubulin served as a loading control. F, quantitative densitometric analysis of PARP levels shown in E. Graph shows the relative amount of PARP in HeLa cells following treatment with the indicated conditions that have been normalized to the amount of γ-tubulin.